Accurate, reliable diagnostic assays for Lyme disease are critical to ensure successful treatment and full recovery. However, serodiagnosis of the disease is particularly difficult due to the high level of genetic heterogeneity of B. burgdorferi isolates, even among those collected from a single location. Furthermore, current serodiagnostic tests for Lyme disease lack sensitivity and specificity for detecting B. burgdorferi infection in the early stages of the disease. To address these deficiencies, we will fabricate protein microarrays based on multiple genospecies of Borrelia spp. for use in developing a sensitive and specific single-tier Lyme disease assay. Using genomic and proteomic approaches, we will first perform comprehensive comparisons between Borrelia genomes to identify unique, membrane associated and/or positively selected antigens of B. burgdorferi sensu stricto. We will then develop proteome microarrays consisting of these individual B. burgdorferi proteins and profile the humoral immune response using sera from B. burgdorferi infected individuals. We hypothesize that differences in the gene content of Borrelia strains will account for the dramatic differences in the antigenic response of human sera in protein arrays that used the B31 isolate as test reference. Our preliminary protein array data using proteins from B. burgdorferi strains B31, N40, JD1 and 297 has already revealed that the sensitivity of our assay far exceeds that of the two commercially-available Lyme disease assays in patients in the early stages of the disease. Thus, a comprehensive investigation of the Borrelia proteome using microarrays will identify the spectrum of antigenic proteins expressed during infection. We will utilize this information to expand upon our array platform to develop a sensitive and specific single-tier Lyme disease assay that will potentially have a wide range of activity and specificity against Lyme disease. This technology can ultimately be adapted to include other Borrelia species found in the USA, Europe and Asia, and used to develop a diagnostic Lyme disease test for world-wide use that may eventually have the potential to include prognostic probability of late symptoms. PUBLIC HEALTH RELEVANCE: Since Lyme disease is a difficult infection to diagnose by the primary care physician, we will develop a new diagnostic test that is able to detect the disease at its onset. By comprehensively identifying the Lyme proteins that the patient recognizes, we will be able to identify the key proteins which all patients respond to when they first come to the doctor's office. This will ultimately allow the development of an office test which will facilitate the quick initiation of therapy which is associated with cure.